The lymphatic vascular system is essential for lipid absorption/transport from the digestive system, maintenance of tissue fluid homeostasis, and immune surveillance. It is also involved in many pathological processes such as inflammatory disease and tumor metastasis, as well as lymphedema, in which the drainage of lymphatic fluid from the interstitial tissue is insufficient, causing the tissues to swell. Lymphedema is most commonly attributed to impaired lymphangiogenesis or lymphatic-valve deficiency; however, the complex nature of lymphatic vessel abnormalities and disease remain poorly understood. Thus, our long-term goal is to understand the molecular and cellular mechanisms responsible for the formation and function of the lymphatic vascular system. FoxC1 and FoxC2 are closely related members of the Fox (Forkhead box) transcription factor family, and our group has previously shown that murine Foxc1 and Foxc2 have numerous essential roles in cardiovascular development. Mutations in human FOXC2 are responsible for the autosomal dominant syndrome Lymphedema-distichiasis, which is characterized by the obstruction of lymphatic drainage in the limbs, venous valve failure, and the growth of an extra set of eyelashes. Recent studies have shown that Foxc2 is also essential for lymphatic valve formation, but the role of FoxC1 in the lymphatic system has yet to be defined. Thus, we have recently generated mice that carry lymphatic endothelial cell (LEC)-specific mutations for Foxc1, Foxc2, or both, which will enable us to determine the precise functions of Foxc1 and Foxc2 in the cellular processes that lead to the development of lymphatic vessels. Our initial studies indicate that the loss of Foxc1 and Foxc2, alone or in combination, leads to lymphatic hyperplasia and is accompanied by declines in the expression of RasGAP genes and by the hyperactivation of ERK. Furthermore, our new genome-wide RNA-seq analysis indicates that Notch signaling genes such as Notch1 and Dll4 are significantly downregulated in LECs that have been isolated from conditional compound LEC-Foxc1;LEC-Foxc2 mutants. Thus, the objective of this application is to study the role of the transcription factors Foxc1 and Foxc2 in lymphatic vessel development. Our central hypothesis is that Foxc1 and Foxc2 are essential for lymphatic cell fate determination, lymphangiogenesis, and lymphatic valve development, and we will test our central hypothesis and accomplish the objective of this application by pursuing the following three Specific Aims: 1) To determine whether Foxc1 and Foxc2 participate in lymphatic cell fate determination by interacting with the Notch signaling pathway. 2) To define the mechanisms by which Foxc1 and Foxc2 regulate the Ras/ERK pathway during lymphangiogenesis. 3) To elucidate the cooperative and distinct functions of Foxc1 and Foxc2 in lymphatic valve development. In summary, the outcome of our project is an extensive characterization of the critical roles that Foxc1 and Foxc2 play in lymphatic vessel development. We expect our findings to have an important positive impact on patient care, because completion of the proposed studies will likely lead to the development and refinement of novel strategies for preventing and treating lymphatic disorders, such as lymphedema.